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WO2024138565A1 - Protéine à nanopores, mutant et utilisation associée - Google Patents

Protéine à nanopores, mutant et utilisation associée Download PDF

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Publication number
WO2024138565A1
WO2024138565A1 PCT/CN2022/143608 CN2022143608W WO2024138565A1 WO 2024138565 A1 WO2024138565 A1 WO 2024138565A1 CN 2022143608 W CN2022143608 W CN 2022143608W WO 2024138565 A1 WO2024138565 A1 WO 2024138565A1
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WIPO (PCT)
Prior art keywords
mutation
nanopore
limited
nanopore protein
nucleotide sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2022/143608
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English (en)
Chinese (zh)
Inventor
王乐乐
刘欢欢
姜楠
王子
陈俊毅
季州翔
郭斐
曾涛
黎宇翔
董宇亮
章文蔚
徐讯
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BGI Shenzhen Co Ltd filed Critical BGI Shenzhen Co Ltd
Priority to PCT/CN2022/143608 priority Critical patent/WO2024138565A1/fr
Priority to CN202280102226.5A priority patent/CN120282978A/zh
Publication of WO2024138565A1 publication Critical patent/WO2024138565A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to the field of nanopore sequencing, and in particular to nanopore proteins and mutants thereof and applications thereof.
  • Nanopore sequencing is a third-generation sequencing technology that has emerged in recent years. It has brought disruptive changes to the gene sequencing industry due to its advantages such as long read length, high throughput, low cost and portability. Nanopore sequencing technology has a wide range of applications in basic theoretical research in life sciences and clinical practice in biomedicine.
  • Nanopore sequencing technology is a sequencing technology based on electrical signals, with Oxford Nanopore Technologies (ONT) as the main representative. This technology can be applied to DNA, RNA and protein sequencing at the same time. It records the electrical signals generated by the continuous blockage when the analytes pass through the nanopore protein one by one in real time, and converts them into sequence information through analysis to achieve sequencing. This technology has high advantages in sequencing speed, throughput, portability, and direct RNA sequencing, and has received widespread attention in recent years.
  • nanopore sequencers such as MinION, GridION and PromethION, as well as commercial instruments such as the QNome-3841 nanopore gene sequencer.
  • the nanopore single-molecule sequencer is a detection system that is highly integrated with multiple disciplines and technologies. The development of this instrument requires deep cross-disciplinary and collaborative innovation in physics, biology, chemistry, semiconductors, computers and other disciplines, and builds a high-precision nanopore single-molecule sequencing system from the underlying core modules.
  • Nanopore sequencing requires that the sensing region inside the pore protein is sharp enough to have high spatial resolution in both the horizontal and vertical directions.
  • pore-forming toxin proteins produced by bacteria or other organisms that destroy the permeability of cell membranes
  • transporter proteins that serve as transport channels for various biological macromolecules and small molecules inside and outside cells
  • viral connectors that provide genome transport channels when viruses infect hosts.
  • MspA Mycobacterium smegmatis pore protein A
  • CsgG curli-specific transport channel
  • the present invention provides nanopore proteins and mutants and applications thereof.
  • the present invention provides a nanopore protein having:
  • amino acid sequences having 70%, 75%, 80%, 85%, 90% or more homology to the amino acid sequence shown in (I) or (II) are also provided.
  • the present invention also provides a mutant of the nanopore protein, comprising:
  • the mutation of the transmembrane region includes any one or more mutations in E166, R200, T204 or S220; and/or
  • the mutation of R200 includes but is not limited to A, G, V, L, I, Y, F or W; and/or
  • the mutation of E116 includes but is not limited to K, R, N, A, G, S, T or Q; and/or
  • the mutation of K124 includes but is not limited to N, A, G, S, T or Q; and/or
  • the present invention also provides a method for preparing the construct, which comprises the following steps:
  • the present invention also provides a biosensor, which includes any of the following items and acceptable auxiliary agents or components:
  • the present invention also provides a single molecule sequencing method, which comprises the following steps:
  • A The nanopore protein according to claim 1; and/or
  • the construction of the sequencing library specifically includes: annealing two partially complementary DNA strands (top strand and bottom strand) to form a linker, connecting the double-stranded target fragment to be tested with T4 DNA ligase at room temperature and purifying to prepare a sequencing library.
  • the sequencing library is then incubated with the helicase BCH105 at 25°C for 1 hour (molar concentration ratio 1:8), and after cross-linking and purification, a sequencing library containing the BCH105 motor protein is formed.
  • the phospholipid bilayer is composed of diacylphosphatidylcholine (DPhPC, 1,2-diphytanoyl-sn-glycero-3-phosphocholine).
  • DPhPC diacylphosphatidylcholine
  • the present invention also provides a sequencing device, comprising the biosensor and an acceptable auxiliary agent or carrier.
  • the present invention found a new type of nanopore protein BCP52 from the deep-sea metagenome. Through protein preparation and nanopore sequencing verification, it was shown that it has the ability to be applied to nanopore sequencing, and the optimization of its mutants can improve the accuracy of nanopore sequencing.
  • Figure 1 shows the three-dimensional structure of BCP52 predicted by Alphafold2 multimer (side view);
  • Figure 2 shows the three-dimensional structure of BCP52 predicted by Alphafold2 multimer (top view);
  • FIG4 is a schematic diagram showing the amino acid residues in the sensor region of the predicted structure of the nanopore protein
  • Figure 6 shows some key amino acids in the entry region of the predicted structure of BCP52
  • the present invention uses a gene mining method of computer-aided structure prediction to mine a nanopore protein from deep-sea metagenomes, so that it can be used as a detection protein and applied to nanopore sequencing, including the detection of small molecules, DNA, RNA and polypeptides.
  • the present invention is verified by protein preparation and nanopore sequencing, indicating that nanopore protein has the ability to be applied to nanopore sequencing, and the optimization of its mutants can improve the accuracy of nanopore sequencing.
  • OD 600 value reaches about 0.6-0.8
  • the bacterial solution was collected by centrifugation at 8000 rpm and the bacteria were frozen at -20°C until use.
  • Buffer A 20mmol/L Tris-HCl, 250mmol/L NaCl, 1% DDM, pH 8.0.
  • the pore opening current of the wild-type nanopore protein (SEQ ID No. 1) and the nanopore protein mutant 1 (SEQ ID No. 2) after being inserted into the phospholipid membrane and applying different voltages was mainly attempted.
  • the current trace of the library DNA passing through the nanopore protein mutant 1 (F80N, SEQ ID No. 2) protein is shown in Figure 11. It can be seen that the nanopore protein mutant 1 can be used for DNA sequencing. As the DNA perforates, the current trace oscillates, and the sequencing amplitude is about 50pA.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Plant Pathology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention relève du domaine du séquençage par nanopores, et concerne en particulier une nouvelle protéine à nanopores BCP52, un mutant et une utilisation associée. Selon la présente invention, une nouvelle protéine à nanopores BCP52 est découverte dans un métagénome provenant des profondeurs marines, la préparation de protéine et la vérification de séquençage par nanopores montrent que la protéine à nanopores BCP52 a la capacité d'être appliquée à un séquençage par nanopores, et l'optimisation du mutant de la protéine à nanopores BCP52 peut améliorer la précision de séquençage par nanopores.
PCT/CN2022/143608 2022-12-29 2022-12-29 Protéine à nanopores, mutant et utilisation associée Ceased WO2024138565A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2022/143608 WO2024138565A1 (fr) 2022-12-29 2022-12-29 Protéine à nanopores, mutant et utilisation associée
CN202280102226.5A CN120282978A (zh) 2022-12-29 2022-12-29 纳米孔蛋白及其突变体和应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/143608 WO2024138565A1 (fr) 2022-12-29 2022-12-29 Protéine à nanopores, mutant et utilisation associée

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WO2024138565A1 true WO2024138565A1 (fr) 2024-07-04

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WO (1) WO2024138565A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011147890A1 (fr) * 2010-05-25 2011-12-01 Vib Vzw Marqueur épitopique pour des applications fondées sur l'affinité
CN108699138A (zh) * 2016-03-02 2018-10-23 牛津纳米孔技术公司 突变孔
CN110621692A (zh) * 2017-05-04 2019-12-27 牛津纳米孔技术公司 由两个CsgG孔组成的跨膜孔
CN110914290A (zh) * 2017-06-30 2020-03-24 弗拉芒区生物技术研究所 新颖蛋白孔
CN113195736A (zh) * 2018-11-08 2021-07-30 牛津纳米孔科技公司 孔

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011147890A1 (fr) * 2010-05-25 2011-12-01 Vib Vzw Marqueur épitopique pour des applications fondées sur l'affinité
CN108699138A (zh) * 2016-03-02 2018-10-23 牛津纳米孔技术公司 突变孔
CN110621692A (zh) * 2017-05-04 2019-12-27 牛津纳米孔技术公司 由两个CsgG孔组成的跨膜孔
CN110914290A (zh) * 2017-06-30 2020-03-24 弗拉芒区生物技术研究所 新颖蛋白孔
CN113195736A (zh) * 2018-11-08 2021-07-30 牛津纳米孔科技公司 孔

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